RNase H activity: structure, specificity, and function in reverse transcription

Abstract

This review compares the well-studied RNase H activities of human immunodeficiency virus, type 1 (HIV-1) and Moloney murine leukemia virus (MoMLV) reverse transcriptases. The RNase H domains of HIV-1 and MoMLV are structurally very similar, with functions assigned to conserved subregions like the RNase H primer grip and the connection subdomain, as well as to distinct features like the C-helix and loop in MoMLV RNase H. Like cellular RNases H, catalysis by the retroviral enzymes appears to involve a two-metal ion mechanism. Unlike cellular RNases H, the retroviral RNases H display three different modes of cleavage: internal, DNA 3' end-directed, and RNA 5' end-directed. All three modes of cleavage appear to have roles in reverse transcription. Nucleotide sequence is an important determinant of cleavage specificity with both enzymes exhibiting a preference for specific nucleotides at discrete positions flanking an internal cleavage site as well as during tRNA primer removal and plus-strand primer generation. RNA 5' end-directed and DNA 3' end-directed cleavages show similar sequence preferences at the positions closest to a cleavage site. A model for how RNase H selects cleavage sites is presented that incorporates both sequence preferences and the concept of a defined window for allowable cleavage from a recessed end. Finally, the RNase H activity of HIV-1 is considered as a target for anti-virals as well as a participant in drug resistance.

Figure 1

Co-crystal structure of HIV-1 reverse transcriptase and an RNA/DNA substrate. A ribbon diagram of the HIV-1 reverse transcriptase associated with a PPT RNA/DNA hybrid, based on the report by Sarafianos et al. (pdb entry 1HYS) (Sarafianos et al., 2001). The polymerase, connection, and RNase H domains of p66 are shown in blue, yellow, and red, respectively; p51 is shown in gray. The RNA template and DNA primer strands are shown in green and purple, respectively. The location of the RNase H active site is indicated with the four key catalytic residues shown as ball and stick structures (Asp443, Glu478, Asp498 and Asp549). The approximate locations of the primer terminus and the RNase H primer grip are also indicated, as is the approximate distance in base pairs between the polymerase and RNase H active sites on the hybrid substrate. Diagram generated with Swiss PDB Viewer.

Figure 2

Schematic representation of the two-metal ion mechanism of catalysis for the RNase H activity of HIV-1 reverse transcriptase [adapted from (Nowotny and Yang, 2006)]. The four acidic residues, Asp443, Glu478, Asp498, and Asp549, in the RNase H active site are drawn in green with the RNA substrate shown in red. The two metal ions are indicated in yellow and labeled as A and B, and metal ion coordination and hydrogen bonds are indicated with dashed lines. The attacking nucleophile is shown in black with an arrow drawn to the scissile phosphate. A key water molecule mediating an interaction between Asp498 and metal ion A is indicated as a blue oval.

Figure 3

The three modes of cleavage for retroviral RNase H. Reverse transcriptase is shown schematically in blue, with the active sites of the RNase H (RnH) and polymerase (Pol) domains indicated. The hybrid substrate contains RNA (red) and DNA (black) strands, with the 3′ ends indicated by an arrowhead. A. DNA 3′ end-directed cleavage. B. RNA 5′ end-directed cleavage. C. Internal cleavage.

Figure 4

The positions and base preferences flanking internal and RNA 5′ end-directed RNase H cleavage sites for HIV-1 and MoMLV reverse transcriptases. The locations of preferred positions relative to a cleavage site at the scissile phosphate (arrow) located between nucleotides −1 and +1 are indicated on an RNA strand (thick black line). The preferences for or against nucleotides for HIV-1 RNase H are shown above the line and for MoMLV RNase H are shown below the line. The preferred nucleotides for each position are indicated in uppercase (strongly preferred) or lowercase (preferred) for internal (blue) and RNA 5′ end-directed (violet) cleavage sites. Disfavored nucleotides for internal cleavage sites are indicated in red.

Figure 5

Comparison of sequences surrounding cleavage sites for the tRNA primer and PPT region of HIV-1 and MoMLV with the positions and preferred nucleotides for internal cleavage. The relevant sequences flanking the PPT and tRNA regions are indicated for HIV-1 (A) or MoMLV (B). The −1/+1 site for cleavage is indicated by an arrow and a vertical line. The match between the preferred positions and an internal cleavage site is indicated as preferred (blue box) or disfavored (red box). The DNA portion of the extended tRNA is underlined. Alignment of the tRNA is shown for the cleavage at the RNA-DNA junction (RNA-DNA) and for cleavage between two ribonucleotides that leaves a ribo A at the end of the minus strand (RNA-riboA).

Figure 6

Selection of cleavage sites by retroviral RNase H. Five different cleavage sites are shown on an RNA strand as A - E with various nucleotide positions numbered as indicated. Four hybrid substrates (thick black lines) are indicated below where a filled circle represents the position of an RNA 5′ or a DNA 3′ recessed end on substrates 1–3 and the blunt end on substrate 4. All sites are eligible for internal cleavage (blue) in substrate 4, where the RNA/DNA hybrid has blunt ends. Otherwise sites on a given substrate are only cleaved if they fall within the window of cleavage for RNA 5′ end-directed (violet) or DNA 3′ end-directed (green) cleavage. For substrate 1 where the recessed end begins at position 1, sites B and C are cleaved by either form of end-directed cleavage and site A is present but not cleaved. For substrate 2 where the recessed end begins at position 6, sites C and D are cleaved if the recessed end is RNA, only site D is cleaved if the recessed is DNA, and sites A and B are present but not cleaved. For substrate 3 where the recessed end begins at position 8, site D is cleaved if the end is RNA, sites D and E are cleaved if the end is DNA, and sites A, B, and C are present but not located within an end-directed window.